Mutation of Tyrosine in the Conserved NPXXY Sequence Leads to Constitutive Phosphorylation and Internalization, but Not Signaling, of the Human B2 Bradykinin Receptor

• Published in: The Journal of biological chemistry Vol. 279; no. 30; pp. 31268 - 31276
• Main Authors: Kalatskaya, Irina, Schüssler, Steffen, Blaukat, Andree, Müller-Esterl, Werner, Jochum, Marianne, Proud, David, Faussner, Alexander
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 23.07.2004
• Subjects: Amino Acid Sequence; Cell Line; Conserved Sequence; GTP-Binding Protein alpha Subunits, Gq-G11 - metabolism; Humans; In Vitro Techniques; Kinetics; Ligands; Mutagenesis, Site-Directed; Phosphorylation; Protein Conformation; Receptor, Bradykinin B2 - chemistry; Receptor, Bradykinin B2 - genetics; Receptor, Bradykinin B2 - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Signal Transduction; Tyrosine - chemistry; Tyrosine - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M401796200

Although the G protein-coupled receptors (GPCRs) share a similar seven-transmembrane domain structure, only a limited number of amino acid residues is conserved in their protein sequences. One of the most highly conserved sequences is the NP XX Y motif located at the cytosolic end of the transmembrane region-7 of many GPCRs, particularly of those belonging to the family of the rhodopsin/β-adrenergic-like receptors. Exchange of Tyr 305 in the corresponding NPLVY sequence of the bradykinin B 2 receptor (B 2 R) for Ala resulted in a mutant, termed Y305A, that internalized [ 3 H]bradykinin (BK) almost as rapidly as the wild-type (wt) B 2 R. However, receptor sequestration of the mutant after stimulation with BK was clearly reduced relative to the wt B 2 R. Confocal fluorescence microscopy revealed that, in contrast to the B 2 R-enhanced green fluorescent protein chimera, the Y305A-enhanced green fluorescent protein chimera was predominantly located intracellularly even in the absence of BK. Two-dimensional phosphopeptide analysis showed that the mutant Y305A constitutively exhibited a phosphorylation pattern similar to that of the BK-stimulated wt B 2 R. Ligand-independent Y305A internalization was demonstrated by the uptake of rhodamine-labeled antibodies directed to a tag sequence at the N terminus of the mutant receptor. Co-immunoprecipitation revealed that Y305A is precoupled to G q/11 without activating the G protein because the basal accumulation rate of inositol phosphate was unchanged as compared with wt B 2 R. We conclude, therefore, that the Y305A mutation of B 2 R induces a receptor conformation which is prone to ligand-independent phosphorylation and internalization. The mutated receptor binds to, but does not activate, its cognate heterotrimeric G protein G q/11 , thereby limiting the extent of ligand-independent receptor internalization.